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The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion...
Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine–Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses...
Condensin activity establishes and maintains mitotic chromosome condensation, however the mechanisms of condensin recognition of specific chromosomal sites remain unknown. rDNA is the chief condensin binding locus in Saccharomyces cerevisiae, and the level of nucleolar transcription is one of the key factors determining condensin loading to the nucleolar organizer. A new aspect of this transcriptional...
The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved...
Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4°C) used to isolate protein...
The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM...
The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like...
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